With the advent of high-throughput screening of small-molecule, nucleic acid, and polypeptide arrays, new challenges have arisen to provide accurate and fast data acquisition. Depending on the type of array, numerous configurations and methods for data acquisition are known in the art.
For example, arrays can be electronically queried to detect hybridization of a nucleic acid sample to an immobilized probe of a probe array, typically allowing an operator to obtain a relatively high number of data in relatively short time. While such systems are advantageous in various respects, several difficulties remain. For example, the quantification range for determination of hybridized molecules is at least in some circumstances less than desirable. Furthermore, if molecules other than nucleic acids need to be determined electronic detection may not be available or technically feasible. Still further, electronic detection is relatively expensive and poses at least some difficulties where a user wants to customize an array.
To circumvent at least some of the disadvantages associated with electronic detection, arrays may be optically queried to detect binding of an analyte to an immobilized probe of a probe array. Most commonly, such arrays have a generally flat surface and optical detection is performed With a scanner analyzing emitted and/or absorbed light from a label that is bound to the analyte. In one typical example, a scanner for such arrays (typically disposed on a microscope slide) may illuminate the array using narrowband excitation (e.g., using a laser), while a photo-multiplier tube (PMT) is used as detector. Narrow band excitation is particularly advantageous since a higher quantum density is achieved at the label, which will generally result in a higher resolution acquisition. Alternatively, the array may be illuminated using wideband excitation (e.g., Xenon light) and a charge coupled device (CCD) detector is employed to detect the signal from the label. Among other advantages, wideband excitation is particularly advantageous where multiple labels are detected using the same scanner.
However, regardless of the particular excitation-type, numerous problems associated with scanner-based detection remain. Most significantly, the illuminated array is frequently not positioned at the same distance relative to the detector (e.g., the array is warped, or has a surface unevenness), typically resulting in inaccurate detection of the signal. Conceptually, such inaccuracies could be resolved by providing a focusing mechanism to the scanner. However, such focusing mechanism would likely significantly reduce the speed at which the scanner would acquire data. Moreover, by exposing non-analyzed analytes to illumination conditions for prolonged periods of time, photobleaching of the labels will most likely result in inaccuracies of the later obtained test results.
To compensate for surface unevenness, spot-by-spot illumination with spot-by-spot analysis can be performed as described in U.S. Pat. No. 6,471,916. Such systems further advantageously allow calibration of signal strength using calibration points on the array. However, while such systems typically provide relatively accurate data, the time required for focusing the detection optics on a spot-by-spot basis increases with increasing array size. Thus, even with relatively small arrays (e.g., 50 to 100 probes), detection time tends to become unacceptable, especially where high-throughput screening is desired.
Thus, although various systems for optical detection of signals from microarrays are known in the art, numerous problems still remain. Therefore, there is still a need for improved configurations for optical microarray detectors.